DESCRIPTION: Cationic antimicrobial peptides, polymyxins along with cryptdins secreted by intestinal epithelial cells and defensins cationic antimicrobial peptides (CAP) found in the granules of mammalian phagocytic cells can be important agents of environmental stress to bacterial pathogens spending time in both natural and host microenvironments. In addition, Salmonella serovars face a variety of other potentially lethal environmental stresses including nutrient starvation. S. typhimurium upon starvation undergo a dramatic physiological alteration, the starvation-stress response (SSR), that protects the cell against the harmful effects of prolonged starvation, as well as providing cross-resistance to other environmental stresses such as oxidative and thermal challenges. This cross-resistance is dependent upon the stress-specific s factor s. Because starved cells, compared to log-phase cells, are generally more resistant to environmental stress, and starvation is a common stress encountered by S. typhimurium in it journeys, we examined the possibility that starved or stationary-phase cells are more resistant to the membrane permeabilizing effects of polymyxin B (PmB), than actively growing log-phase cells. We recently reported that starved/(LB)-stationary-phase-cells exhibit ~200 to 1500-fold increase cross-resistance to a 60 minute PmB challenge as compared to log-phase cells. This PmB cross-resistance was found to involve both phoP-dependent and phoP-independent pathways. Furthermore, both pathways are independent of ss indicating that they are different from other known ss-dependent cross-resistance mechanisms. Both pathways are important for PmB resistance early during C-starvation and LB-stationary-phase. However, only the phoP-independent pathway is important for P-starvation induced PmB resistance and the sustained PmB resistance seen in 24 hour C-starvation or LB-stationary-phase cells. Recent studies suggest that the phoP-independent pathway involves the pmrA locus. The specific aims of this proposal are designed to begin the genetic, molecular and biochemical characterization of these novel starvation-/stationary-phase-inducible PmB resistance pathways. This will be accomplished by (1) isolating mutants altered in one or more of these PmB cross-resistance mechanisms by insertional mutagenesis, (2) cloning and partial sequencing of the chromosomal DNA adjacent to insertion junction sites in an effort to determine the possible functions, and (3) the assessment of LPS and whole cell protein patterns at different times during starvation and LB-stationary-phase and in different mutant backgrounds.